Mutation of human mu opioid receptor extracellular "disulfide cysteine'' residues alters ligand binding but does not prevent receptor targeting to the cell plasma membrane

The FL opioid receptor, a primary site of action in the brain for opioid ne uropeptides and opiate drugs of abuse, is a member of the seven transmembra ne, G protein-coupled receptor (GPCR) superfamily. Two cysteine residues, o ne in each of the first two of three extracellular loops (ECLs), are highly conserved among GPCRs, and there is direct or circumstantial evidence that the residues form a disulfide bond in many of these receptors. Such a bond would dramatically govern the topology of the ECLs, and possibly affect th e position of the membrane-spanning domains. Recent findings from several l aboratories indicate the importance of the ECLs for opioid ligand selectivi ty. These conserved cysteine residues in the mu opioid receptor were studie d using site-directed mutagenesis. Little or no specific binding of radiola bled opiate alkaloid or opioid peptide agonists or antagonists was observed for receptors mutated at either "disulfide cysteine" residue. Each mutant mu opioid receptor was expressed in both transiently- and stably-transfecte d cells, in some cases at levels comparable to the wild type receptor. The two point mutants possessing serine-for-cysteine substitutions were also ob served to successfully reach the cell plasma membrane, as evidenced by elec tron microscopy. Consistent with related work with other GPCRs, the mu opio id receptor apparently also employs the extracellular disulfide bond. This information now permits accurate molecular modeling of extracellular aspect s of the receptor, including plausible scenarios of mu receptor docking of opioid Ligands known to require specific extracellular loop features for hi gh affinity binding. (C) 1999 Elsevier Science B.V. All rights reserved.